The purpose of this proposal is to investigate how intracellular free calcium (Ca2+) in airway smooth muscles is regulated by membrane potential and by neurotransmitters and related agents. This proposal will address these questions by directly measuring intracellular Ca2+. Although the importance of intracellular (Ca2+) in the control of contractile state of smooth muscle is well accepted, little direct evidence exists of the nature of the changes in Ca2+ and the role played by membrane potential in the action of neurotransmitters and other agents which affect the contractile state of the muscle. These studies will utilize a recently developed double barrelled Ca2+-selective microelectrode to record changes in intracellular Ca activity and membrane potential in response to acetylcholine, histamine, isoproterenol and other agents which produce changes in the contractile state of airway smooth muscle. This evidence will establish the relationships between changes in membrane potential and changes in intracellular Ca2+. For these studies we shall employ single, freshly isolated smooth muscle cells obtained from bovine trachealis muscle. The studies will utilize isolated cells to overcome complications inherent in intact tissues such as the presence of neural elements, heterogeneous population of cell types and the mechanical and electrical coupling between cells. These studies should establish the activity of Ca2+ in resting cells, the effects of contractile agents on Ca2+, the effects of relaxing agents on Ca2+, the effects of these agents on membrane potential and the relationship between membrane potential and alterations in Ca. From these studies there should emerge a basic understanding of the regulation of intracellular Ca2+ in airway smooth muscles, which will undoubtedly bridge further steps in elucidating hyperexcitability of some disease state such as asthma.